Intended use

This immunoassay kit allows for the use in vitro quantitative determination of human Beta-Endorphin, β-EP concentrations in cell culture supernates, serum, plasma and other biological fluids.

 

Introduction

Endorphins are endogenous opioid biochemical compounds. They are polypeptides produced by the pituitary gland and the hypothalamus in vertebrates, and they resemble the opiates in their abilities to produce analgesia and a sense of well-being. In other words, they might work as "natural pain killers." Using drugs may increase the effects of the endorphins. Beta-Endorphin is an endorphin produced by the pituitary gland that is a potent pain suppressant.

Beta-endorphin is released into the blood (from the pituitary gland) and into the spinal cord and brain from hypothalamic neurons. The beta-endorphin that is released into the blood cannot enter the brain in large quantities because of the blood-brain barrier. The physiological importance of the beta-endorphin that can be measured in the blood is far from clear: beta-endorphin is a cleavage product of POMC which is the precursor hormone for adrenocorticotrophic hormone (ACTH), so it will be released whenever ACTH is released. The behavioural effects of beta-endorphin are exerted by its actions in the brain and spinal cord, and probably the hypothalamic neurons are the major source of beta-endorphin at these sites. In situations where the level of ACTH is increased (e.g. Addison disease), the level of endorphins also increases slightly.

Beta-endorphin has the highest affinity for the μ1-opioid receptor, slightly lower affinity for the μ2- and δ-opioid receptors and low affinity for the κ1-opioid receptors. μ-receptors are the main receptor through which morphine acts. Classically, μ-receptors are presynaptic, and inhibit neurotransmitter release; through this mechanism, they inhibit the release of the inhibitory neurotransmitter GABA, and disinhibit the dopamine pathways, causing more dopamine to be released. By hijacking this process, exogenous opioids cause inappropriate dopamine release, and lead to aberrant synaptic plasticity which causes addiction. Opioid receptors have many other and more important roles in the brain and periphery however, modulating pain, cardiac, gastric and vascular function as well as possibly panic and satiation, and receptors are often found at postsynaptic locations as well as presynaptically.

 

Test principle

The microtiter plate provided in this kit has been pre-coated with an antibody specific to β-EP. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for β-EP and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB (3,3'5, 5' tetramethyl-benzidine) substrate solution is added to each well. Only those wells that contain β-EP, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of β-EP in the samples is then determined by comparing the O.D. of the samples to the standard curve.